1. Field of the Invention
The present invention relates to a confocal microscope which excites a sample labeled with a fluorescence dyestuff and a fluorescent protein by use of an excitation wavelength, and detects a fluorescence emitted from the sample.
2. Description of the Related Art
Heretofore, as a confocal microscope, a multi-color confocal microscope in which a multi-wavelength fluorescence detection system is employed has been known (e.g., see U.S. Pat. No. 5,127,730).
The multi-color confocal microscope irradiates a sample having positions differently dyed with two or more fluorescent colorants with laser beams having wavelengths corresponding to the respective fluorescent colorants, and then detects fluorescent wavelengths, at which excitation occurs to generate the fluorescence, through wavelength separating means such as a dichroic mirror for these fluorescent wavelengths.
A confocal microscope capable of estimating a polarizing direction of the fluorescence dyestuff has also been known. For example, in the confocal microscope disclosed in Jpn. Pat. Appln. KOKAI Publication No. 8-254654, a sample is irradiated with laser beams emitted from a laser light source through an objective lens, and then through the objective lens, the fluorescence emitted from the sample is branched into two optical paths by the dichroic mirror. Afterward, the branched beams are passed through polarizers which cross with each other at right angles, to obtain two images in accordance with the polarization of the fluorescence dyestuff.
In recent years, for example, when a target protein of living cells is labeled with a fluorescence to observe distribution or movement thereof, a fluorescent protein such as GFP (green fluorescent protein) has often been utilized as a marker tracer.
A sample labeled with such GFP has polarization properties as described in BIOPHOTONICS International May, 2002, p. 10. Thus, by detecting the fluorescence polarization of the sample using a fluorescent protein such as GFP, it becomes possible to analyze molecular movement of the protein and a fluorescent life. Moreover, a molecular structure of the fluorescent protein changes by light stimulation, chemical reaction, electrical stimulation, pH and temperature variation or the like, so that the polarization properties change. Therefore, the analysis of the polarization properties of the fluorescent protein enables the analysis of a function of the protein.
Additionally, in the U.S. Pat. No. 5,127,730, only a general detecting method of a multi-wavelength fluorescence is disclosed, and the detection of a fluorescence having polarized components is not described. Moreover, in Jpn. Pat. Appln. KOKAI Publication No. 8-254654, it is described that two images are acquired in accordance with polarization of a fluorescence dyestuff by the fluorescence from the sample passed through the polarizers which cross with each other at right angles, but only the confocal microscope which emits a wavelength light to generate the fluorescence is described. Moreover, an excitation method for obtaining the fluorescence having the polarized components with the use of the fluorescent proteins such as GFP as the fluorescence label is not described in the Jpn. Pat. Appln. KOKAI Publication No. 8-254654.